The pH is a measurement of the acidic or alkaline nature of an aqueous substance. Said substance can exhibit greatly varying properties depending on the current pH thereof. For example, in the case of blood, the pH affects the oxygen-binding properties of blood pigments, such as haemoglobin or haemocyanin. The lower the pH, the less oxygen the blood pigment can bind via the prosthetic group thereof (i.e. iron or copper). This property makes it easier to release oxygen in the tissue when the pH of the blood there lowers due to metabolic release of carbon dioxide (the Bohr effect). If, conversely, carbon dioxide is exhaled via the lungs, the pH of the blood there rises and thus the ability of the blood pigment to absorb oxygen increases. Changes in pH influence the oxygen-binding curves which show the relationship between the prevailing oxygen partial pressure and the percentage of oxygen which is bound to the blood pigment (blood pigment saturation). The oxygen gradient as a decrease in the oxygen partial pressures between the ambient air and the cells of the body determines the absorption of oxygen. Optimal oxygen absorption and release allows efficient oxygen transport and thus makes it possible for the organism to perform very efficiently. Oxygen-rich blood changes shade owing to changes in the conformation of each prosthetic group when binding oxygen. For example, oxygenated haemoglobin exhibits a brighter and more vibrant shade than deoxygenated haemoglobin, whereas oxygenated haemocyanin turns blue. This leads to a change in the absorption behaviour of the substance sample when it is irradiated with ultraviolet or visible light. By simultaneously measuring the pH and the absorption behaviour to determine the oxygen-binding properties of blood in the case of in vivo or almost in vivo studies, important information relating to the current state of the substance or the carrier thereof can thus be obtained. In this case, however, it is also important in particular to be able to manage with only small sample volumes of the substance to be studied, since the amount of the sample material is often very limited (for example in the case of minute organisms).
U.S. Pat. No. 5,564,419 A discloses a device for determining photometrically in vitro the oxygen content in a blood sample. By means of a syringe-type device, the blood can be drawn directly out of the organism and conducted via a channel. Said channel is passed through by openings in a carrier, through which openings light is shone to measure absorption. The openings are sealed with a sealing ring. However, a pH measurement is not carried out. EP 2 110 430 B1 discloses a measuring cell for measuring the biochemical oxygen demand, in which cell the end of a fibre-optic cable from a spectrophotometer is brought into contact with the substance to be studied via a sealing element. U.S. Pat. No. 5,056,520 discloses a needle-shaped optical measuring sensor comprising a fibre-optic cable made from silicon, by means of which sensor the pH of a blood sample can be determined. The fibre-optic cable is guided by a sleeve and the measuring tip thereof ends in a drop of the substance to be studied.
The publication “Method for Continuous Registration of O2-Binding Curves of hemoproteins by Means of a Diffusion Chamber” by H. Sick and K. Gersonde (in Analytical Biochemistry 32, 362-376 (1969) discloses a diffusion chamber for photometrically measuring the absorption behaviour of blood under various gas exposures. In a central cylinder, the blood sample to be studied is placed on an acrylic glass slide and is irradiated with light from a mercury vapour lamp which is detected in a photodetector. The acrylic glass slide is sealed with respect to the central cylinder by a sealing ring. The gas mixtures are introduced into the central cylinder via a gas supply. A pH measurement is not carried out.
The publication “Oxygen evolution in a hypersaline crust: in situ photosynthesis quantification by microelectrode profiling and use of planar optode spots in incubation chambers” by J. Woelfel et al. (in Aquat Microb Ecol Vol. 56:263-273, 2009) discloses using an oxygen microoptode comprising sensor spots and fibre optics for optically measuring the primary production of oxygen in the Arctic. In this case, the oxygen generation of ground-dwelling diatoms outdoors in a benthic chamber is detected online via optical fibres. The basic principle of optical oxygen measurement is based on exciting a specific fluorescent dye (indicator molecule) using light of a defined wavelength and quenching said dye according to the oxygen concentration; see URL cited Jun. 20, 2013 http://www.angewandteoekologie.uni-rostock.de/en/forschung3/analytik0/mikroopt/.
The publication “Temperature Effects on Hemocyanin Oxygen Binding in an Antartic Cephalopod” by S. Zielinski et al. (in Biol. Bull. 200:67-76 (February 2001) describes a diffusion chamber which comprises a modified quartz glass cuvette for simultaneously determining changes in pH and absorption of blood of a cephalopod under in vitro conditions. When it is added to the cuvette, the blood to be studied (400 μl) is distributed to an upper and lower reservoir, which is continuously mixed by means of magnetic stirrers. In a region between the two reservoirs, the blood additionally disperses into a thin layer (0.45 mm) in which the absorption measurement takes place. The measuring tip of a micro pH electrode is introduced into the blood sample via an upper sealing cover to measure the pH. A second opening in the cover makes it possible to expose the sample to a gas mixture which can be varied during the measurement (oxygen, carbon dioxide, nitrogen). The change in absorption which occurs is measured using a spectrophotometer comprising glass-fibre optics. The precise construction is not shown in greater detail in the publication; see FIG. 1 and the paragraph “Analysis of oxygen binding”.
However, the measuring procedure is described in detail. The analysis of the oxygen saturation and binding properties of blood pigments by measuring light absorption is predominantly carried out using blood samples which have been buffered to stabilise the pH. Buffers are aqueous solutions of a weak acid and the conjugated base thereof. When adding acidic (having H3O+ ions) and alkaline solutions (OH), the hydronium ions of the buffer base and the hydroxide ions of the buffer acid are quenched (buffered) so that the pH changes only a little during said addition. However, a buffer is only effective if relatively large amounts of buffer acid and buffer base are present. However, the buffering has a demonstrable effect on the binding properties of the blood pigment and thus does not always accurately reflect the actual physiological properties. In the case of Pörtner (1990 (“An analysis of the effects of pH on oxygen binding by squid (lllex illecebrosus, Loligo pealei) haemocyanin”, J. Exp. Biol. 150, 407), 1994 (“Coordination of metabolism, acid-base regulation and haemocyanin function in cephalopods”, Mar. Fresh. Behav. Physiol. 25, 131-148)) and Zielinski et al. (2001, “Temperature effects on hemocyanin oxygen binding in an Antarctic cephalopod”, Biol. Bull. (Woods Hole) 200, 67-76), this problem is solved in that native, unbuffered blood was studied at a constant oxygen partial pressure, but with a varied pH. By measuring additional binding curves at various oxygen partial pressures, the same parameters could thus be determined as in the case of conventional methods having the above-mentioned disadvantages. However, in the case of the method of Pörtner and Zielinski et al., the inventors have recognized that it is disadvantageous that high sample consumption occurs and the pH measurements are carried out on pooled blood samples, and therefore the measurements cannot be clearly attributed to individual organisms and also cannot be statistically evaluated.